Comp. Biochem. Physiol. Vol. 119B, No. 1, pp. 201–211, 1998 ISSN 0305-0491/98/$19.00Copyright 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(97)00308-8

Pertussis Toxin-Sensitive GTP-Binding ProteinsCharacterized in Synaptosomal Fractions of Embryonic

Avian Cerebral CortexGeorge G. Holz1 and Timothy J. Turner2

1Diabetes Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, U.S.A.; and2Department of Physiology, Tufts University School of Medicine, Boston, MA 02111, U.S.A.

ABSTRACT. Pertussis toxin (PTX)-sensitive GTP-binding proteins (G proteins) are essential intermediariessubserving neuronal signal transduction pathways that regulate excitation-secretion coupling. Despite this estab-lished role, relatively little is known regarding the identity, subcellular distribution, and relative abundance ofthis class of G proteins in synaptic nerve endings. Here, sucrose density gradient centrifugation was combinedwith 1- and 2-dimensional gel electrophoresis to characterize PTX-sensitive G protein α subunits in synaptosomalfractions of embryonic (day 12) chick cerebral cortical homogenates. These findings demonstrate multiple iso-forms of Mr 40–41 kDa Giα and Goα subunits that can be identified on the basis of PTX-catalyzed ADP-ribosyla-tion and immunoblot analysis. comp biochem physiol 119B;1:201–211, 1998. 1998 Elsevier Science Inc.

KEY WORDS. G protein, pertussis toxin, neuron, synapse

INTRODUCTION the C-terminus of G protein α subunits. It is this catalyticaction of the S1 subunit that blocks activation of G proteins

Pertussis toxin (PTX), a bacterial exotoxin secreted by viru-by cell surface receptors (38).lent strains of Bordetella pertussis, is a unique pharmacologi-

In vertebrate nervous systems it is the α subunits ofcal probe for analysis of signal transduction pathways medi-

Gj and Go proteins that serve as substrates for PTX (5,28,ated by heterotrimeric guanyl nucleotide-binding proteins 29,42,46). These Mr 39–41 kDa α subunits (Giα and Goα,[G proteins; (9,40,53)]. PTX blocks diverse G protein-medi-

respectively) are structurally homologous, yet functionallyated phenomena, including inhibition of adenylyl cyclase,

distinct guanyl nucleotide-binding proteins with intrin-stimulation of phospholipases A2 and C, modulation of ion sic GTP-ase activity. To date, three distinct subtypes of Giαchannel gating, and regulation of stimulus-secretion cou-

and multiple subtypes of Goα have been described. All arepling. This broad spectrum of physiological antagonism re-

members of a large family of high molecular weight G pro-sults from PTX-catalyzed ADP-ribosylation, a covalent teins that includes transducin (Gt, found in retinal photore-modification of G protein α subunits that prevents their

ceptors) and the PTX-insensitive G proteins Gs, and Golf,activation by cell surface receptors. Notably, the specificityand Gz (31,34). In neurons, the PTX-sensitive G proteins

with which PTX targets G proteins has allowed a direct are essential components subserving signalling pathwaysassessment of the role that these signal transducing ele-

that regulate excitation-secretion coupling (1,12,21,22,55).ments play in synaptic plasticity, differentiation, and gene

For example, PTX was reported to block alpha-2 adrenergic,expression.

GABA-B, and opiate receptor-mediated inhibition of cal-PTX is one member of a family of structurally related ‘‘A–

cium channel function (21), as well as the stimulatory effectB’’ toxins that also includes cholera and diptheria toxins

of acetylcholine on potassium channel function (41). Such(16,48). The S1 subunit of PTX is the active (A) protomereffects correlate positively with the ability of PTX to block

which catalyzes mono-ADP-ribosylation of G proteinsinhibitory effects of noradrenaline, GABA, and enkephalin

through formation of a thioglycosidic bond between ADP-on the Ca21-dependent exocytosis of noradrenaline (1),

ribose (derived from NAD1) and the sulfhydryl moiety ofsubstance P (13,22), and glutamate (12) from synaptic

a consensus cysteine residue located four amino acids fromnerve endings. In general, the importance of the Giα andGoα PTX substrates to processes regulating synaptic function

Address reprint requests to: George G. Holz, Wellman 320, Massachusetts has remained somewhat enigmatic due, in part, to uncer-General Hospital, 50 Blossom Street, Boston, MA 02114, U.S.A. Tel. 617- tainties concerning their subcellular localization in nerve726-5191; Fax 617-726-6954; E-mail: holz@helix.mgh.harvard.edu

terminals. Here is reported the characterization of PTX-sen-Received 14 July 1997; revised 15 September 1997; accepted 18 Septem-ber 1997. sitive Giα and Goα proteins in synaptic nerve endings of em-

202 G. G. Holz et al.

Preactivation of PTXbryonic (day 12) chick cerebral cortex. These findingsshould provide a foundation for future studies directed at PTX (List Biochemicals) was preactivated (30 min, 21°C)determining the developmental regulation of G protein ex- in distilled H2O containing (in mM): 100 Tris HCl (pHpression as it relates to synaptic function in the central ner- 7.8), 25 dithiothreitol, and 1 ATP. The activated PTX wasvous system. stored at 220°C in 50% glycerol. ATP minimizes direct

effects of guanyl nucleotides on the enzymatic activity ofPTX by saturating the nucleotide recognition site on theMATERIALS AND METHODStoxin.(33,35,39).Preparation of Cerebral Cortical Homogenates

Freshly dissected cortices obtained from 12-day-old chickADP-Ribosylation of G Proteinsembryos were suspended in ice-cold Buffer A containing (in

mM): 100 Tris HCl (pH 7.8), 1.2 MgCl2, 0.2 EDTA, 1.5 Table 1 lists the composition of Solutions 1–3 used in theEGTA, 16 dithiothreitol, and (in mg/ml) 5.0 d-glucose, 0.1 ADP-ribosylation reaction. Preactivated PTX was added toleupeptin, 0.1 soybean trypsin inhibitor. Tissue was homog- ice-cold Solution 2 prior to initiating the ADP-ribosylationenized on ice in a Wheaton C homogenizer, and the homog- reaction. The reaction was then initiated by adding Solu-enate was centrifuged (20 min, 200 3 g; 4°C) to obtain a tion 2 to substrate proteins to yield 50 µl of Solution 3 con-P1 pellet. The supernatant was then recentrifuged (20 min, taining PTX (final concentration 2.4 µg/ml, unless other-100,000 3 g; 4°C) to obtain a P2 pellet and a soluble frac- wise noted), and (in mM): 85 Tris HCl (pH 7.8), 10tion (cytosol). P1 and P2 preparations were washed twice in dithiothreitol, 10 thymidine, 10 isoniazide, 6 MgCl2, 3ice-cold Buffer A. The pellets were then resuspended in ATP, 0.9 EGTA and (in µM): 100 GTP, 120 EDTA, 5Buffer A containing the indicated concentration of deter- NAD and 0.5 [32P]NAD (New England Nuclear NEG-023,gent (typically 0.1% Lubrol). Proteins were solubilized by a 0.7–1.3 µCi/assay, final specific activity 1.9–4.7 Ci/mmol).second round of homogenization to yield a final preparation Typically, the ADP-ribosylation reaction was allowed tocontaining 6–9 mg protein/ml, as determined by the proceed for 1 hr at room temperature (21°C).method of Peterson (45). For experiments examining theelectrophoretic mobility of G proteins (Figs. 1–3) or charac-

Pretreatment of Homogenates with Guanyl Nucleotidesteristics of the ADP-ribosylation reaction (Figs. 4, 5–7, and8), particulate material in detergent-solubilized homoge- Homogenates solubilized in Lubrol and suspended in Buffernates was removed by centrifugation (15 min, 3,600 3 g; A were treated for 30 min with guanyl nucleotides at the4°C) prior to initiating the ADP-ribosylation reaction. In indicated temperatures. The ADP-ribosylation reaction wascontrast, quantitative ADP-ribosylation and immunoblot- then initiated by adding Solution 2 to the sample proteins.ting of subcellular fractions (Table 2; Fig. 6) was assessed Although no attempt was made to vary the duration of pre-using homogenates that were not precleared by centrifuga- treatment, it is recognized that the rate at which guanyltion, thereby avoiding removal of less readily soluble pro- nucleotides associate with G proteins is dependent on theteins that might serve as substrates for PTX. subtype of α subunit, the temperature, and the concentra-

tion of added MgCl2.(8,15). GTP, GTP-γ-S, Gpp(NH)p,and Gpp(CH2)p (lithium salts) were from Boehringer-

Subcellular Fractionation Mannheim.Discontinuous density gradient centrifugation of cerebralcortical homogenates was performed according to Whitta-

Quantification of PTX-Catalyzed ADP-Ribosylationker et al. (54). All procedures were conducted at 4°C. Corti-ces were homogenized in 0.32 M sucrose (10% w/v), centri- PTX-catalyzed incorporation of [32P]ADP-ribose was deter-

mined by liquid scintillation counting of trichloroaceticfuged (10 min, 1,000 3 g), and the pellet discarded. Thesupernatant (S1) was centrifuged (20 min, 10,000 3 g), the acid (TCA)-precipitated proteins. After incubation with

PTX in sealed 10 3 75 mm borosilicate glass culture tubes,resulting supernatant (S2) discarded, and the pellet (synap-tosomal fraction) collected. The synaptosomal fraction was the reaction was terminated by adding 1 ml of 0.03% (w/v)

sodium deoxycholate solution. Protein and carrier detergentsuspended in lysis buffer (10 mM Tris HCl, pH 7.8; 1 mMEDTA), equilibrated for 1 hr, and centrifuged (20 min, were precipitated with 100 µl of 72% TCA (w/v), vortexed,

and centrifuged (3,600 3 g, 15 min). The supernatant was10,000 3 g). The pellet was discarded and the supernatantwas collected to obtain Fraction A. Fraction A was layered aspirated carefully and the pellet was resuspended in 1 ml

0.1 N NaOH. After vortexing, solubilized proteins were re-on a sucrose step gradient (0.4–1.2 M, 0.2 M increments)and centrifuged (2 hr, 53,000 3 g). Gradient fractions (4 precipitated with 100 µl 72% TCA, and centrifuged a sec-

ond time (3,600 3 g, 15 min). The supernatant was aspi-ml each) were collected, diluted twofold, and recentrifuged(60 min, 200,000 3 g). The resulting pellets were then re- rated and the pellet resuspended in 0.5 ml of a solution

containing two parts dH2O, one part 10% SDS (w/v), andsuspended in buffer A.

PTX Substrates in Avian Cerebral Cortex 203

TABLE 1. The chemical composition of solutions included in the ADP-ribosylation assay. Sample protein was suspended inhomogenization buffer, and PTX was preactivated as described in Materials and Methods. All solutions were prepared on iceimmediately prior to initiating the ADP-ribosylation reaction

Solution 1 Solution 2 Solution 3

ConcentrationIngredient (mM) Ingredient Volume (ml) Ingredient Volume (ml)

MgCl2 60 Solution 1 50 Solution 2 20NAD 0.05 PTX (60 µg/ml) 20 Sample Protein 30ATP 30 Tris HCl (100 mM) 110Isoniazide 120 [32P] NAD (1 µCi) 20Thymidine 100GTP 1.0Tris HCl (pH 7.8) 13EDTA 0.2EGTA 1.5Dithiothreitol 16

one part 0.8 N NaOH. 32P-incorporation was measured by was determined by measuring the pH of slices obtained fromblank tube gels run in parallel with each set of samples, andliquid scintillation counting. With this protocol $90% of

[32P]ADP-ribose incorporation was associated with proteins was linear between pH 4.5 and 6.5. Gels were equilibratedin 2% SDS (w/v), 10% glycerol, 5% 2-mercaptoethanol,of Mr 40–41 kDa, as confirmed by scintillation counting of

SDS-PAGE gel slices. and 62.5 mM Tris HCl (pH 6.8) prior to electrophoresis inthe second dimension by discontinuous SDS-PAGE.

SDS-PAGE of Ribosylated ProteinsImmunoblot Analysis of Pertussis Toxin SubstratesRibosylated proteins were diluted 1:1 in sample buffer con-

taining 187.5 mM Tris HCl (pH 6.8), 5.0% (v/v) 2-mercap- Primary antisera tested included affinity-purified prepara-tions of AS/7 (18), directed against the C-terminus deca-toethanol and (in % w/v): 30% sucrose, 3.0% SDS, and

.003% bromophenol blue. Following denaturation of the peptide sequence (KENLKDCGLF) of mammalian trans-ducin α, and GO/1 (19) directed against the C-terminussamples (100°C, 5 min), discontinuous sodium dodecyl sul-

fate polyacrylamide gel electrophoresis (SDS-PAGE) was sequence (ANNLRGCGLY) of mammalian Goα. AS/7, butnot GO/1, cross-reacts with mammalian Giα 1 and 2. GO/performed according to Laemmli (32). Polyacrylamide

stacking (4% T) and resolving (10% T) gels contained bis- 1 recognizes Go α subunits purified from bovine brain, andexhibits weak cross-reactivity with mammalian Giα3. Immu-acrylamide (2.5% C), and were run at constant current (35

mA/gel, 3.5 hr) in a water-cooled vertical electrophoresis noblotting was performed as described by Goldsmith et al.(18). Following SDS-PAGE, proteins were electro-trans-unit. Proteins were visualized by Coomassie blue (14) or

silver staining (37). 14C-methylated reference standards ferred (30 V, 16 hr) onto nitrocellulose membranes (0.45µm). Electrophoresis was confirmed by noting transfer onto(Amersham) included (in kDa): phosphorylase b, 92.5; bo-

vine serum albumin, 69.0; ovalbumin; 46.0; carbonic anhy- the nitrocellulose of prestained molecular weight markers,or by silver staining of gels subsequent to transfer. Nitrocel-drase, 30.0; and lysozyme, 14.3 Mr was determined by a plot

of Rf vs. log molecular weight. Gels were dried and used to lulose membranes were exposed to a 500-fold dilution ofthe primary antiserum overnight, followed by a 2-hr incuba-expose Kodak X-Omat AR film (0.5–14 days, 270°C) with

Cronex intensifying screens. tion in HRP-conjugated, goat anti-rabbit secondary antise-rum (Kirkegaard and Perry). Immunoreactivity was visual-ized using 4-chloro-napthol dye solution as substrate for

Analysis by Isoelectric Focusingperoxidase.

Isoelectric focusing (IEF) was performed according to theAmes and Nikaido (2) variation of the O’Farrell (44) proce-

Immunoprecipitation of G Proteinsdure. Ribosylated proteins were diluted 1:2 in sample buffercontaining 9.5 M urea, 5% (v/v) 2-mercaptoethanol, and Lubrol-solubilized P2 membranes (10 µg protein in 50 µl

Buffer A) were radiolabeled by PTX-catalyzed ADP-ribosy-(in w/v) 1.6% LKB ampholine pH 5–7, 0.4% LKB ampho-line pH 3.5–10.0, 8% NP-40, and 1% SDS. Samples were lation. The radiolabeled proteins were added to 130 µl

NMT buffer containing (in mM): 150 NaCl, 10 MgCl2, andapplied to the basic end of IEF tube gels (4% T, 2.5% Cpolyacrylamide, 1.5 mm diameter), and electrophoresed 20 Tris HCl (pH 7.8). Affinity-purified G protein antiserum

(final concentration 1–80 µg/ml) was then added, and the(500 V, 16 hr) without prefocusing. The IEF pH gradient

204 G. G. Holz et al.

preparations was assessed by SDS-PAGE/autoradiography.As illustrated in lanes 1 and 2 of Fig. 1, specific labeling(defined as that requiring PTX) was resolved as a faint 40/41 kDa doublet (lower arrow), whereas non-specific labelingof a 115 kDa substrate was commonly observed (upperarrow). Comparison of lanes 1 and 3 illustrates that the in-tensity of specific labeling was dramatically increased by sol-ubilization of the P2 preparations in 0.1% Lubrol prior toinitiating the ADP-ribosylation reaction. In contrast, label-ing of the 115 kDa substrate remained unaffected (cf., lanes2 and 4). Under these conditions of detergent solubilization,the unidentified substrate for non-specific labeling remainedin the particulate fraction and was readily eliminated fromthe assay by centrifugation (cf., lanes 3 and 5; see Materialsand Methods). Lubrol-solubilization and centrifugationtherefore provide a means by which specific labeling is opti-mized in this assay (cf., lanes 1 and 5).

Detection of ADP-Ribosylated Gi and GoFIG. 1. Optimization of PTX-catalyzed ADP-ribosylation.

SDS-PAGE combined with autoradiography and immu-Cerebral cortical proteins were resolved by SDS-PAGE, andan autoradiogram of the dried gel was intentionally overex- noblotting was performed to assess whether the Mr 40/41posed to illustrate the method by which selective labeling kDa PTX substrates in P2 homogenates were in fact α sub-was achieved. In a P2 homogenate not solubilized with Lu- units of Gi and Go. As illustrated in Fig. 2 ribosylated pro-brol, specific incorporation appeared as a faint doublet

teins were resolved on 10% gels as a clearly defined doublet(lower arrow, lane 1) running at ca. 40 kDa, whereas non-specific labeling appeared as a single band running at ca. 115kDa (upper arrow, lanes 1,2). The PTX-independent non-specific labeling most likely reflects the activity of endoge-nous poly ADP-ribosyltransferases. Note that when the ho-mogenate was solubilized in 0.1% Lubrol, specific labelingwas markedly enhanced (lane 3), whereas non-specific la-beling was unaffected (lane 4). High speed centrifugation(airfuge) of the solubilized preparation to remove the 115kDa substrate eliminated nonspecific labeling (lanes 5 and6). All ADP-ribosylation reactions were run in parallel, andeach lane on the gel received 40 mg of membrane protein.

mixture was allowed to equilibrate overnight at 4°C. Im-mune complexes were specifically adsorbed by incubation(4 hr, 4°C) with Protein G Sepharose CL4B (PharmaciaLKB, equilibrated in NMT-buffer). Absorbed immune com-plexes were pelleted by centrifugation (3,600 3 g, 5 min), FIG. 2. SDS-PAGE combined with autoradiography and im-

munoblotting identifies Gi and Go proteins serving as sub-washed twice in NMT-buffer, resuspended in scintillant,strates for PTX-catalyzed ADP-ribosylation. (Left) Autora-and counted.diogram of a 10% gel illustrating the relative electrophoreticmobility of ribosylated G proteins in a P2 homogenate (20mg protein). [14C]-methylated molecular weight standardsRESULTS(lane 1) served as reference markers for estimating Mr. In-PTX-Catalyzed ADP-Ribosylation of Cerebral Corticalcorporation of [32P]ADP-ribose appeared as a Mr 40 and 41G ProteinskDa doublet (lane 2). (Right) Immunoblot of ribosylatedproteins (100 mg/lane) performed with antisera that distin-We sought to identify assay conditions under which PTXguish between Gia (AS/7) and Goa (GO/1). P2 homogenatecatalyzed the selective incorporation of [32P]ADP-ribose inproteins were resolved by SDS-PAGE and transferred to ni-Goα and Giα substrates. Figure 1 illustrates the protocol withtrocellulose for immunostaining. Note that GO/1 immuno-

which such selectivity was achieved. Cerebral cortical ho- reactivity appeared as a single 40 kDa band (lane 1), whereasmogenates were fractionated by differential centrifugation, AS/7 immunoreactivity appeared as a 40 and 41 kDa dou-

blet (lane 2).whereupon ADP-ribosylation of crude synaptosomal (P2)

PTX Substrates in Avian Cerebral Cortex 205

(left), and then transferred to nitrocellulose for immunoblot third protein corresponds to pI 6.0 and Mr 41 kDa. Thetendency of these proteins to aggregate (and streak) whenanalysis (right). Primary antisera tested included AS/7 and

GO/1, exhibiting specificity for Giα and Goα, respectively. loaded on IEF tube gels in sufficient concentration to allowimmunological detection precluded immunoblot analysis ofWhen nitrocellulose blots were probed with these antisera,

three forms of immunoreactivity were detected. AS/7 la- the 2-D gels. However, the overall pattern of radiolabelingsuggests that the predominant 40 kDa acidic substrate isbeled a 40/41 kDa doublet (Giα-like immunoreactivity),

whereas GO/1 labeled a single 40 kDa band (Goα-like) that most likely Goα, whereas the three more basic 40/41 kDasubstrates may correspond to isoforms of Giα (see Discus-apparently co-migrated with the smaller form of Giα. Auto-

radiograms prepared from both blots revealed radiolabeled sion).proteins migrating as a doublet with electrophoretic mobil-ity identical to that of the immunoreactive labeling (data

Quantification of PTX-Catalyzed ADP-Ribosylationnot shown). This was to be expected since these antiserarecognize not only native, but also ribosylated forms of α Since G proteins constitute the predominant substrate for

PTX in these cortical membranes, quantitative measure-subunits.ments of ADP-ribosylation were made possible by scintilla-tion counting of TCA-precipitated samples. To optimize

Multiple Isoforms of Gi and Go this assay, a systematic analysis was performed examiningTwo-dimensional gel electrophoresis combining isoelectric the time-course, temperature-dependence, and detergent-focusing and SDS-PAGE was performed to test whether the sensitivity of the ADP-ribosylation reaction. As illustrateddoublets observed on autoradiograms of 1-D gels result from in Fig. 4A, specific incorporation (defined as [32P]ADP-ri-radiolabeling of three distinct α subunits, as suggested by bose incorporation observed in the presence of PTX minusthe immunoblot analysis. Figure 3 illustrates an autoradio- that in its absence) approached an equilibrium value (45gram prepared from a 2-D gel demonstrating not only three, pmol/mg pro) within 120 min following addition of PTXbut four or more distinct substrates for PTX-catalyzed ADP- to the reaction mix. Incorporation was linearly related toribosylation in these P2 homogenates. The predominant protein concentration (4A, inset), and as illustrated in Fig.substrate is a relatively acidic protein (pI 5.4) of Mr 40 kDa 4B, increasing concentrations of PTX accelerated the rateand constitutes ca. 55% of total substrate, as determined by but not the extent of labeling. In P1 homogenates (4A),densitometry. Also evident are three more basic proteins, this equilibrium value was ca. 25 times that observed in theeach accounting for ca. 15% of total labeling. Two share a absence of added PTX. In contrast, in P2 homogenates (4B),similar pI (5.8) but differ in Mr (40 vs 41 kDa), whereas a a 40–60-fold stimulation was observed, reflecting the differ-

ential enrichment of substrate in these two preparations(see below Table 2A).

PTX-catalyzed ADP-ribosylation also exhibited signifi-cant temperature-dependence. As illustrated in Fig. 5A, therate but not the extent of specific incorporation was de-creased by lowering the reaction temperature. T1/2 (the timerequired to reach one-half the equilibrium value of incorpo-ration) was 9, 13, and 24 min at 37, 21, and 4°C, respec-tively. Lower incubation temperatures are preferred sincethermal denaturation of the substrate was readily observed(see below).

A similar quantitative analysis was performed to charac-terize in greater detail the detergent-sensitivity of the ADP-ribosylation reaction. As illustrated in Fig. 5B, homogenatesFIG. 3. Two-dimensional analysis of ribosylated G proteins

by combined IEF/SDS-PAGE/autoradiography. A P2 ho- were assayed for PTX-catalyzed [32P]ADP-ribose incorpora-mogenate (1 mg) was applied to the basic end of a 4% poly- tion under conditions of non-ionic (Lubrol, Tween-20, Tri-acrylamide tube gel for IEF (500 V, 16 hr) in the first dimen-

ton X-100, and NP-40), zwitterionic (CHAPS), or anionicsion (arrow 1). Separation in the second dimension (arrow(deoxycholate) detergent solubilization. Consistent with a2) was by discontinuous SDS-PAGE (10% resolving gel). Il-

lustrated is an autoradiogram of the 2-D gel demonstrating previous report that detergent-solubilization directly stimu-four distinct substrates for PTX-catalyzed ADP-ribosylation. lates the enzymatic activity of PTX (39), a 9.7-, 12.8-, andNote that the predominant substrate is a relatively acidic 13.6-fold stimulation of specific incorporation relative toprotein (pI 5.4) of Mr 40 kDa. Note also three more basic

control (i.e., no detergent) was observed following solubili-proteins, two of which share similar pIs (5.8) but which dif-zation in 0.1% Lubrol, NP-40, and Triton X-100, respec-fer in Mr (40 vs. 41 kDa). A third protein corresponds to pI

6.0 and Mr 41 kDa. tively. CHAPS, Tween-20, and deoxycholate were less ef-

206 G. G. Holz et al.

2B, ADP-ribosylation of the individual step gradient frac-tions demonstrated that significant quantities of PTX sub-strate reside in fractions 1–5 (0.4–1.2 M sucrose), with frac-tions 1 and 2 containing ca. 57% of the total. Fractionationin this manner allowed full recovery of Giα and Goα: ca. 96%of the applied substrate retained its activity in the subse-quent ADP-ribosylation assay. The accuracy with whichthis assay distinguishes amongst the individual fractions wasconfirmed by SDS-PAGE/immunoblot analysis. As illus-trated in Fig. 6, a wide-spread but differential distributionof PTX substrates was noted, visualized by AS/7 and GO/1 immunostaining. Note that the overall pattern of immu-nostaining (Fig. 6) is in agreement with the pattern of[32P]ADP-ribose incorporation observed in the individualgradient fractions (Table 2).

Guanyl Nucleotide-G Protein Interactions Analyzed byADP-Ribosylation

Guanyl nucleotides regulate the conformational state of Gproteins, thereby determining the efficiency with which αsubunits serve as substrates for PTX. To assess whether thisADP-ribosylation assay provides an analytical tool withwhich to analyze these interactions, two conformation-de-pendent processes were examined: stabilization of G pro-

FIG. 4. (A) PTX-catalyzed ADP-ribosylation is saturable teins by GTP, and their activation by GTP-γ-S. Figure 7with time and is a linear function of protein concentration. summarizes experiments that demonstrate a role for guanylIllustrated is the time course of [32P]ADP-ribose incorpora-

nucleotides in protecting G proteins against thermal dena-tion in a P1 homogenate (90 mg protein/assay, 21°C).turation. As illustrated in Fig. 7A, homogenates preincu-Squares and triangles denote specific (1120 ng PTX/assay)

and non-specific (2PTX) incorporation, respectively. Inset bated at 4 or 21°C prior to addition of PTX retained theirillustrates the protein concentration-dependence of ADP-ri- ability to be ADP-ribosylated irrespective of whether thebosylation (100 min incubation). A linear regression line preincubate contained added GTP. In contrast, incorpora-was fit to the data (r2 5 0.987). (B) Increasing concentra-

tion of [32P]ADP-ribose was nearly eliminated by preincuba-tions of PTX (30–240 ng/50 ml assay) accelerate the ratetion at 37°C in buffer to which no GTP was added. Notably,but not the extent of ADP-ribosylation (P2 homogenate; 80

mg protein per assay). Numerical values in A and B are ex- thermal denaturation was blocked by including 100 µMpressed as the mean value for each determination (n 5 3). GTP in the preincubate, whereas 100 µM GTP-γ-S pro-The standard error of the mean was #5% for each determi- vided only partial protection.nation in this and in all subsequent figures.

Figure 7B illustrates that the stabilizing action of GTPwas mediated by a high affinity binding site (EC50, 8 µM)that also recognized GDP-β-S. Also tested were GTP-γ-S,fective in descending order. Increasing the concentrationGpp(NH)p, and Gpp(CH2)p, analogs of GTP that activateof all six detergents to 1% resulted in a generalized inhibi-G proteins. Compared to GTP and GDP-β-S, these analogstion of the ADP-ribosylation reaction (data not shown).exhibited ca. 25-fold greater potency in the assay (EC50 forGTP-γ-S, 0.3 µM). This observation is as expected since

Subcellular Distribution of Gi and Go the affinity of the nucleotide binding site for activating nu-cleotides is known to exceed that for GTP (24). Note alsoThe relative abundance of PTX substrates in various subcel-

lular fractions was assessed using cerebral cortical homoge- that the efficacy of these activating analogs (defined as max-imal protective action) was consistently less than that mea-nates fractionated by differential centrifugation. As summa-

rized in Table 2A, ca. 62% of the total pool of cerebral sured for GTP: saturating concentrations (10 µM) of GTP-γ-S, Gpp(NH)p, and Gpp(CH2)p were, respectively, 31, 35,cortical PTX substrate resides in the crude synaptosomal

fraction. To more accurately assess the subcellular distribu- and 58% as effective as a saturating concentration of GTP(300 mM). As shown below, the limited efficacy of thesetion of Giα and Goα, we performed sucrose density gradient

centrifugation of an enriched synaptosomal lysate prepared analogs is only apparent: their full protective action ismasked by their activating function.according to Whittaker et al. (54). As summarized in Table

PTX Substrates in Avian Cerebral Cortex 207

TABLE 2. (A) Quantification of PTX-catalyzed ADP-ribosylation in soluble and particulate fractions of chick cerebral cortex.Homogenates were fractionated as described in Materials and Methods, solubilized in 0.1% lubrol, and assayed for incorpora-tion of [32P]ADP-ribose by scintillation counting. The relative distribution of protein in this homogenate was P1 (37.9%), P2

(29.7%), soluble protein (32.4%). (B) Subcellular distribution of PTX substrates as analyzed by discontinuous sucrose densitygradient centrifugation of ADP-ribosylated chick cerebral cortical P2 homogenates. Homogenates (7.5 mg protein) were lysed,fractionated on sucrose step gradients (0.4–1.2 M, 0.2 M increments), solubilized in 0.1% Lubrol, and assayed for PTX-cata-lyzed ADP-ribosylation by liquid scintillation counting. The relative distribution of protein in this preparation was 0.4M(24.7%), 0.6M (15.2%), 0.8M (18.7%), 1.0M (14.4%), 1.2M (7.8%), pellet (19.2%). This is the same preparation as illustratedin Fig. 8

A. PTX-catalyzed incorporation of [32P]ADP-ribose in embryonic chick cerebral cortical homogenates

[32P]ADP-ribose [32P]ADP-ribose incorporationFraction (pmol/mg protein) (% total pmol in all 3 fractions)

P1 (200 3 g) 30 33.7P2 (100,000 3 g) 70 61.6Soluble protein 5 4.7

B. The subcellular distribution of ribosylated G proteins as analyzed by discontinuous sucrose density gradientcentrifugation of cerebral cortical P2 homogenates

Gradient fraction [32P]ADP-ribose [32P]ADP-ribose incorporation(Molarity) (pmol/mg pro) (% total P2 substrate activity)

0.4 100 340.6 108 220.8 84 211.0 63 121.2 24 2Pellet 20 5

% of total P2 substrate activity recovered: 96

Receptor-Site Analysis of the Guanyl Nucleotide Hill equation (B/Bmax) 5 [(S)η/(IC50)η 1 (S)η] where B isBinding Domain [32P]ADP-ribose incorporation, S is nucleotide concentra-

tion, η is the Hill coefficient, and IC50 is the concentrationG proteins no longer serve as efficient substrates for PTX(300 nM) of GTP-γ-S that reduced to half-maximal the nu-when pretreated with activating nucleotides such as GTP-cleotide-sensitive [32P]ADP-ribose incorporation. As illus-γ-S (35,36). As illustrated in Fig. 8A, this interaction be-trated in Fig. 8B, a Hill plot revealed that for GTP-γ-S,tween receptor (G protein) and ligand (GTP-γ-S) wasthe binding interaction exhibited a Hill coefficient of 1.02.quantitated by establishing the dose-dependence of the re-Moreover, as illustrated in Fig. 8C, a plot of fractional re-sponse (inhibition of ADP-ribosylation). Notably, thesponse (B/Bmax) vs. nucleotide concentration confirmedthreshold (0.03 µM) and IC50 (0.3 µM) values for GTP-γ-that for GTP-γ-S, experimentally-derived values for inhibi-S-induced inhibition of ADP-ribosylation matched thetion of ADP-ribosylation match calculated values predictedthreshold and EC50 values for protection by GTP-γ-Sby the Hill equation assuming a η of 1.02.against denaturation (cf., Figs 7B and 8A). This overlapping

concentration-dependence appears to allow GTP-γ-S toprotect G proteins against thermal denaturation while si-

DISCUSSIONmultaneously inducing activation of their α subunits. Thisobservation may explain why the efficacy of GTP-γ-S in the The ADP-ribosylation of cerebral cortical homogenates re-

ported here demonstrates an enrichment of PTX substratesdenaturation assay did not match that of GTP.The IC50 for GTP-γ-S-induced inhibition of ADP-ribosy- in synaptosomal preparations, with significant labeling ob-

served in vesicular and plasma membrane density gradientlation deduced from Fig. 8A exceeds by ca. 10-fold the ap-parent Kd for [35S]GTP-γ-S binding to purified Gi or Go (24). fractions. The substrate specificity of this labeling was con-

firmed by two-dimensional gel electrophoretic analysisAlthough this most likely reflects nonequilibrium bindingof the nucleotide to its receptor under our assay conditions, which revealed selective ADP-ribosylation of four distinct

G protein α subunits. Although definitive characterizationwe sought additional confirmation that GTP-γ-S acts via asingle high affinity binding site. Concentration-response of these substrates will require primary amino acid sequence

information, they are provisionally identified as isoforms ofdata presented in Fig. 8A was modeled according to the

208 G. G. Holz et al.

FIG. 6. Subcellular distribution of Mr 40/41 kDa Gia and Goa-like immunoreactivity in cerebral cortical synaptosomes. P2

lysates were layered on sucrose step gradients (0.4–1.2 M,0.2 M increments) as illustrated, fractionated by discontinu-FIG. 5. (A) Temperature-dependence of ADP-Ribosylation.ous sucrose density gradient centrifugation, and the frac-Illustrated is the time course of [32P]ADP-ribose incorpora-tions assayed for AS/7 (left) or GO/1 (right) immunoreactiv-tion in P1 homogenates incubated at 4, 21, or 37°C as indi-ity by combined SDS-PAGE/immunoblotting (200 mgcated in the inset (68 mg protein/assay). (B) ADP-ribosyla-protein/lane; arrows indicate the direction of electrophore-tion is stimulated by detergent-solubilization. All sixsis). This is the same preparation as analyzed in Table 2B.detergents were tested at a final concentration of 0.1% (aMorphological characterization of this type of lysate byconcentration that exceeds the CMC for all four non-ionicWhittaker et al. (54) demonstrated that the 0.4 M gradientdetergents, but not that for deoxycholate or CHAPS).fraction is enriched in synaptosomes, whereas the 0.6 M[32P]ADP-ribose incorporation was determined after solubili-fraction contains predominantly microsomes.zation and centrifugation to eliminate unsolubilized proteins

(P1 homogenate, 40 mg protein, and 120 ng PTX per assay,21°C). Each data point is the average value of three individ-

tein, accounting for ca. two-thirds of the total substrateual assay determinations.available for ADP-ribosylation in the cortex. Assuming aMr of 40 kDa for α subunits, PTX substrate accounts for0.3–0.4% (w/w) of cellular protein. Although this value isGiα (possibly subtypes 1–3) and Goα on the basis of Mr, pI,in agreement with previous estimates indicating an unex-and cross-reactivity with antisera specific for mammalian αpectedly high concentration of G proteins in the brain, itsubunits. It is noteworthy that mammalian cerebral cortexmay still represent an underestimate. ADP-ribosylation ofalso contains multiple substrates for PTX, including Giα 1purified α subunits by PTX requires addition of βγ dimersand 2 (4,27) and novel forms of Goα (19,23,26,47). Al-to the assay mixture (10,30,42). Such a requirement for βγthough the distribution of these isoforms within nerve ter-might render α subunits unavailable for ADP-ribosylationminals remains largely unexplored, PTX substrates arein cellular lysates (25), thus preventing a direct measure-found in synaptic plasma membranes (7,11,49–51) and se-ment of the total substrate pool. An additional potentialcretory vesicles (43,51).source of error in the assay results from limitations of co-factor availability. Endogenous NAD in cellular lysates may

Limitations of the ADP-Ribosylation Assay dilute the specific activity of radiolabeled NAD, whereasNAD-glycohydrolases may decrease the concentration ofAccurate measurement of PTX substrates in cellular lysates

is complicated by uncertainties regarding the extent to added tracer. We have attempted to minimize these compli-cations by washing membranes with homogenization buffer,which substrate and/or co-factor availability limits the

ADP-ribosylation reaction. In this assay, unfractionated and by including the glycohydrolase inhibitor isoniazide(17) in the reaction mixture.synaptosomes contained 75–100 pmol substrate/mg pro-

PTX Substrates in Avian Cerebral Cortex 209

FIG. 7. Guanyl nucleotides regulate G protein stability. (A)Thermal denaturation of G proteins. P2 homogenates (80 mgprotein/assay) were preincubated for 30 min at 4, 21, or37°C in buffer containing no added nucleotides and either100 mM GTP or GTP-g-S. ADP-ribosylation was then initi-ated by addition of PTX (120 ng/assay, reaction run for 75min at 21°C). Specific incorporation is expressed relative tocontrol (defined as incorporation measured without the pre-incubation step). Note that in the absence of added nucleo-tide, raising the preincubation temperature to 37°C reduced[32P]ADP-ribose incorporation to 10% of control, and thatGTP blocked this effect. (B) Guanyl nucleotides differen-tially protect against denaturation. ADP-ribosylation wasmeasured as described in part A following preincubation for FIG. 8. Receptor-site analysis of guanyl nucleotide-G protein30 min at 37°C in homogenization buffer containing 0.01– interactions. (A) Activating nucleotides inhibit ADP-ribosy-300 mM of GTP, GDP-b-S, Gpp(CH2 )p, Gpp(NH)p, or lation. Homogenates were pretreated (30 min, 21°C) withGTP-g-S (86 mg protein, 120 ng PTX/assay, P2 homogenate, 0.01–300 mM of the indicated nucleotide prior to addition75 min, 21°C). Each data point is the average value of three of PTX to the assay (120 ng PTX/assay, P2 homogenate, 80individual assay determinations. mg protein, reaction run 75 min at 21°C). (B) Hill plot of

experimentally-derived values for GTP-g-S-induced inhibi-tion of ADP-ribosylation. Solid line was obtained by linearregression analysis of the raw data (R2 5 0.989). (C) Concen-Nucleotide Regulation of ADP-Ribosylationtration-response relationship for GTP-g-S-induced inhibi-

We have illustrated the applicability of ADP-ribosylation tion of ADP-ribosylation. Filled circles indicate experimen-for studies directed at analysis of the guanyl nucleotide tally-derived values for fractional response vs. nucleotide

concentration. Solid curve was calculated according to thebinding site central to G protein activation. PretreatmentHill equation (see text). Note that the x-axis in Figs 8B andof cortical homogenates with nucleotides (GTP-γ-S,C refers to the concentration of GTP-g-S added to eachGpp(NH)p, Gpp(CH2)p) that activate G proteins renderedassay. Each data point is the average value of three individ-α subunits insensitive to subsequent ADP-ribosylation by ual assay determinations.

preactivated PTX. In contrast, activating and non-activat-ing nucleotides (GTP-γ-S, GDP-β-S) also protected againstthermal denaturation of the substrate proteins. A likely ex-

210 G. G. Holz et al.

A.G. Purification and properties of the inhibitory guanine nu-planation for these findings is that guanyl nucleotides in-cleotide-binding regulatory component of adenylate cyclase.fluence substrate availability by regulating the conforma-J. Biol. Chem. 259(6):3560–3567;1984.

tional state of G proteins (6,35,36,52). Our concentration- 7. Brabet, P.; Pantaloni, C.; Rouot, B.; Toutant, M.; Garcia-response and receptor-site analyses support this conclusion Sainz, A.; Bockaert, J.; Homburger, V. Multiple species and

isoforms of Bordetella pertussis toxin substrates. Biochem. Bi-and indicate that a single nucleotide recognition site onophys. Res. Comm. 152(3):1185–1192;1988.the α subunit confers not only thermal stability, but also

8. Carty, D.J.; Padrell, E.; Codina, J.; Birnbaumer, L.; Hilde-susceptibility to activation. The half-saturating concentra-brandt, J.D.; Iyengar, R. Distinct guanine nucleotide binding

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9. Carty, D.J. Pertussis toxin-catalyzed ADP-ribosylation. Meth-lation, and binding data for activation by GTP-γ-S was wellods Enzymol. 237:63–70;1994.fit assuming a Hill co-efficient of 1.0. GDP-β-S apparently

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switch necessary to convert the heterotrimer from PTX-sen- talytic support of ADP-ribosylation of alpha subunits by per-sitive to insensitive, as is observed with activating nucleo- tussis toxin but differential interactions with Gs alpha. Bio-

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